Showing papers in "Journal of Plant Biotechnology in 2002"

Mass Production of Paclitaxel by Plant Cell Culture

Abstract: Samyang Genex succeeded in commercialization of anticancer agent-paclitaxel by plant cell culture technology. The core technology of Samyang Genex relating paclitaxel production includes cell line development, cell line preservation, cell culture, scale-up technology, and purification technology. On the basis of the research, Samyang Genex built the factory operated by CGMP (current good manufacturing practice). The paclitaxel-Genexol-is commercially available in Korea, and it will be launched to world market including USA after approval of US FDA.

Micropropagation of Juvenile and Mature Tree of Corylopsis coreana by Axillary Bud Culture

Abstract: We have developed an in vitro micropropagation system via shoot formation from axillary buds using nodal segments of Corylopsis coreana. Explants from both juvenile tree (one-year-old greenhouse stock seedlings) and mature tree (ten-years-old tree in nursery) were compared with regard to propagation efficiency. Combined treatment of both BA and zeatin were effective on shoot proliferation since the best result was obtained on MS medium supplemented with 0.5∼3.0 mg/L zeatin and 0.2 mg/L BA. Generally, juvenile explants were better in both shoot proliferation and growth than mature explants. However, as the duration of in vitro culture was proceed to 6 months, explants from mature tree also produced three shoots per explant. Distinctive differences in rooting and adaptability to soil of shoots obtained from mother trees. Whereas shoots originated from juvenile explants rooted as high as 97%, those from adult explants showed 62% rooting. Similar result was also observed in soil acclimatization. The plantlets derived from juvenile plants survived 67%, while only 48% of those from adult trees survived. The results showed a possibility of the micropropagation of Corylopsis coreana through shoot formation from axillary buds. In addition, the advance of the research still remain to enhance the frequency of acclimatization of plantlets from mature trees for practical application.

Investigation of Transformation Efficiency of Rice Using Agrobacterium tumefaciens and High Transformation of GPAT (glycerol-3-phosphate acyltransferase) Gene Relative to Chilling Tolerance

Abstract: This study has been focused on improving transformation efficiency of rice using Agrobacterium tumefaciens. We have demonstrated the effect of this system when the GPAT gene related to the cold-resistance was transferred by Agrobacterium tumefaciens in rice. Transformation conditions were modified using intron -glucuronidase (GUS) expression as a reporter gene in the rice. In this study, mature seed-derived calli of rice (Oruza sativa L. cv. Dongjin) were pre-cultured for 3 days and then infected with Agrobacterium. When this infected calli were cultured in the dark for 10 days on co-cu]lure medium containing 50 mg/L of CaCl, 30 mg/L of acetosyringone, 2 mg/L of 2,4-D, 120 mg/L of betaine, high GUS expression was observed. In the present transformation system, the efficiency of transformation of GPAT gene was about 54%. Stable integration of GPAT gene into chromosomal DNA was proven by southern blot analysis of genomic DNA isolated from T progenies. The progenies (T1 generation) derived from primary transformant of 5 lines were segregated with a 3 (resistant) : 1 (sensitive ratio) in medium containing hygromycin. This high frequency transformation system can be used as a useful tool in transformation of another monocotyledon.n.

Mass Propagation of Liriodendron tulipifera L. via Somatic Embryogenesis

Abstract: Mass propagation of tulip tree (Liriodendron tulipifera L) via somatic embryogenesis was successfully achieved with immature samaras collected from adult trees Embryogenic tissues were induced by culturing them samaras on 1/2 LM medium (Litvay`s) containing 2,4-D and BA Somatic embryos developed from the embryogenic tissues and germinated to normal plants (emblings) upon transfer onto the same medium containing either AgNO or activated charcoal So far, several factors appeared to influence both the induction of embryogenic tissues and germination of the embryos into plants These include the collection time of samaras for the induction of embryogenic tissue, sucrose level in the culture medium, the level of both AgNO and activated charcoal, and plating density of somatic embryos on germination medium for maturation and germination of somatic embryos into plantlets

Plant Regeneration of Major Cultivars of Sweetpotato (Ipomoea batatas) in Korea via Somatic Embryogenesis

Abstract: An efficient plant regeneration system of major cultivars of sweetpotato (Ipomoea batatas (L.) Lam.) in Korea via somatic embryogenesis was established. Embryogenic calli were formed from shoot apical meristems of sweetpotato cultivars when cultured on LS medium supplemented with 1 mg/L auxin (2,4-D, picloram, dicamba). Among three kinds of auxin, 1 mg/L 2,4-D showed the highest embryogenic calli induction rate. After 4 weeks of cultures on LS medium supplemented with 1 mg/L 2,4-D, embryogenic calli induction rates of Sinhwangmi, Zami, Yulmi, and White Star were 86%, 78%, 76%, and 80%, respectively. Upon transfer onto LS basal medium, most of somatic embryos developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to mature plants in a greenhouse.

Screening of soybean recombinant inbred lines for high competence somatic embryogenesis

Abstract: Cotyledonary explants from immature zygotic embryos of each 85 recombinant inbred lines (RILs) were cultured on medium containing MS salts, B5 vitamins, 40 mg 2,4-dichlorophenoxyacetic acid and 30 g sucrose. Frequency of somatic embryo formation on cotyledonary explants showed in thirty-six lines(＜10%), in thirty-seven lines (11~49%), in nine lines (50~89%), and in three lines(>90%), respectively, The highest frequency (up to 90%) and number (6.36 per cotyledon) of somatic embryos were obtained from lines of KM1010, KM1032 and KM1064. Primary somatic embryos produced from three lines produced numerous secondary somatic embryos on the surfaces, which were subcultured for over one year. Upon transfer to maturation and conversion medium (Komatsuda, 1992), somatic embryos converted to plantlets at a frequency of approximately 25%.

Plant Regeneration and Effect of Auxin and Cytokinin on Adventitious Shoot Formation from Seedling Explant of Taraxacum platycarpum

Abstract: Taraxacum platycarpum has been used as a medicinal plant. We investigated optimal condition for efficient plant regeneration through adventitious shoot formation on medium with various kinds of growth regulators. Adventitious shoot formation was achieved when cytokinin was used alone. Shoot formation was higher on MS medium containing 2 mg/L BAP compared to that with 2 mg/L kinetin and 2 mg/L 2-ip. Among root, hypocotyl and cotyledon, roots were the best explant for the adventitious shoot induction. Adventitious shoot formation from roots declined markedly by the combination of both 0.1 mg/L NAA and 2 mg/L BAP, while shoot formation from cotyledons was stimulated by the same combination. Root formation from the regenerated shoots was achieved on 1/3MS medium containing 0.2 mg/L NAA. Regenerated plantlets was acclimatized and transplanted to the soil, showing 100% survival.

Effect of Plant Growth Regulators on Callus Induction and Plant Regeration of Farfugium japonica

Abstract: The leaf and petiole segments of Farfugium japonica were cultured to investigate the influence of growth regulators on their callus induction and plant regeneration. The callus induction and growth showed a good response both leaf and petiole on MS media supplemented with 1∼2 mg/L 2,4-D and 1∼2 mg/L BA. Callus induction and growth were more effective in petiole segments than leaf one. The highest percentage of plant regeneration was obtained from 60-day-old calli on MS medium supplemented with 1 mg/L NAA and 2 mg/L BA. When subcultured to the same medium for about 60 days, multiple shoots were developed from regenerating callus. The shoots produced roots after transferring to rooting medium containing 0.5 mg/L IAA. The plantlets over 50 mm in height were successfully acclimatized in vermiculite, and the survival rate was over 95%.

Tolerance to Potato Soft Rot Disease in Transgenic Potato Expressing Soybean Ferritin Gene

Abstract: Ferritin is ubiquitous in bacteria, animals and plants. Ferritin is thought to play two main roles in living cells to provide iron for the synthesis of iron protein such as ferretoxin and cytochromes and to prevent damage from radicals produced by iron/dioxygen interaction. To enhance the resistance of potato to Erwinia carotovora, the soybean ferritin gene was introduced into the potato either under CaMV 35S or hsr203J promoter. Potato transgenic plants were screened by PCR analysis using specific primers to the ferritin gene. Expression of ferritin gene under CaMV 35S and hsr203J promoter in potato transgenic plants was confirmed by northern blot analysis. hsr203J promoter known to pathogen inducible in tobacco drives the induction upon Phytophthora infestan in potato and the transcript level of ferritin gene was extremely high after 24 hours post inoculation. One of transformants under CaMV 35S promoter was increased 2.5 fold than untransformant. Each one of transgenic potato containing gene promoter CaMV 35S and hsr203J-ferrtin fusion exhibited tolerance against potato soft rot.

Introduction of VP6 Gene into Potato Plant by Agrobacterium-mediated Transformation and Analysis of VP6 Expression in Transgenic Potatoes

Abstract: A VP6 fragments was subcloned with BamHI in the binary pMBP-1 vector under Califlower Mosaic Virus (CaMV) 355 promoter and neomycin phosphotransferase II (npt II) gene. The recombinant binary vector was mobilized into Agrobacterium-tumefaciens LBA4404 by the freeze-thaw method and potato (Solanum tubensum L. cv Desiree) was transformed by modified leaf-disc cocultivation. Shoots were induced on MS medium with 0.01 mg/L NAA, 0.1 mg/L GA, 2.0 mg/L Zeatin, 100.0 mg/L kanamycin, 500.0 mg/L carbenicillin. In order to identify the copy number of VP6 into potato plant, total genomic DNA was isolated from transgenic potato and analysed by Southern blotting. Genomic DNA and total mRNA analysis demonstrated the incorporation of the foreign gene into the potato genome, as well as their transcription.

Commercial Production of Seed Garlic by Tissue Culture Technique

Abstract: We, Tong Yang Moolsan Co. Ltd. (TYM) set up the mass-production system for virus-free seed garlic via tissue culture technique. TYM`s tissue culture technique is called as `Multiple shoot propagation technique` This technique can lead mass propagation of genetically homogeneous seed garlic in a short period because of its highly proliferation rate of in vitro shoots (15/sup 10//year). TYM researchers applied the technique to some selected garlic cultivars with superior characteristics and carried out field test of productivity in the inside and outside of the country for several years. According to the yearly results of field test with virus-free seed garlic, we ascertained that virus-free seed garlic can produce the highly yield increase (max. above 50%) and also can enhance the product quality. Consequently, we estimated that TYM`s seed garlic will contribute to farmers with increase of income and can elevate the national position of garlic market in the world for its competitive power of technical and production cost.

Evaluation of Morphological Characteristics and RAPD Analysis in Korean Landraces of Naked Barley

Abstract: Barley varieties collected from 1940 to 1951 allover the Korean peninsula by Dr. Takahashi Ryuhei were reintroduced from the Research Institute for Bioresouces in Okayama University, Japan, and the evaluation of morphological characteristics and RAPD analysis were performed. Totally, 493 varieties of Korean barley landraces were planted in the green house, from which seeds of 491 varieties were harvested and conserved in the seedbank of the Rural Development Administration. Majority of the naked barley varieties showed dense spikes with long awn, late heading, winter habits, and long plant height. However, variants having various phenotypes such as short awn, blue aleurone color, brachytic type and waxyness were also identified. Plant height, spike length, and cold-tolerance in the varieties were also highly variable among them. Homogeneity tests on the variation of growth habits, spike density, anthocyanin pigmentation on the seed coat, and hairiness on leaf sheath between naked and covered barley showed that the variations of naked barley were similar to those of covered barley. It maybe indicate that the most of naked barley landraces were mutated from the covered barley landraces. Korean landraces of naked barley were broadly divided into 4 groups by the dendrogram produced by morphological characteristics; however, the identities of the group were rather indistinct. Many varieties, belonged to the same group, were showed different band patterns in RAPD analysis using 5 different primer sets. These results indicate that the 112 varieties of naked barley landraces were different genotypes.

Determination of the Period of the Formation and Size of Sieve Element Area and Sieve Pore

Abstract: During the phloem development from parenchyma cells in a suspension culture of Streptanthus induced sucrose carrier and glucose carrier disappeared. Sieve element area and sieve pore induced suspension culture of Streptanthus were formed almost at the last period of the synthesis of sieve endoplasmic reticulum (SER) and p-protein. The new synthesized cell wall begann to digeste only after the new cell wall was surrounded by SER. The digested region of the cell wall and the formed region of sieve pore were regular comparatively. The completed sieve pore was an oval form, and the outer portion of sieve pore varied, ca 1.2 ~1.6 in longitudinal, 0.8 ~1.3 in tangential, and the inner size of sieve pore was irregular form of a star-like shape. The number of sieve pore between sieve cells was ca 2~7 per and the sieve pore wall with callose was 0.05 ~0.07 in thickness. The energy for the formation of sieve element area and sieve pore might be supplied by mitochondria near the new cell wall and the role of SER remains to be illucidated.

Primer for the Potato Specific Internal Control DNA and Screening Method for the Genetically Modified Potatoes by Competitive Duplex-PCR

Abstract: We report the new method for the screening of genetically modified potato by competitive duplex-PCR using the potato specific single oligomer primer for the internal control and CaMV 35S promoter or NOS terminator specific primers. The single oligomer primer (rAGU4A) amplify the potato specific internal control band from the homozygous potato genomic DNA in the RAPD profiles of all analyzed potato varieties. The 530 bp internal control DNA was amplified independently to CaMV 35S promoter or NOS terminator DNA and identified as repetitive or microsatellite DNA of potato (AF541972). With this new technique, the transgenic potatoes which were transformed with vectors contained the different foreign genes are analyzed. In case of the commercialized transgenic potato varieties, `Hew Leafs`, those two genetic factors are used for promoter and terminator respectively So, this new PCR technique should be a promising method of cost effective and accurate screening for the commercialized GM potatoes on market.

Proteome Data Analysis of Hairy Root of Panax ginseng : Use of Expressed Sequence Tag Data of Ginseng for the Protein Identification

Abstract: For the hairy root of Panax ginseng, we have got mass spectrums from MALDI/TOF/MS analysis and Tandem mass spectrums from ESI/Q-TOF/MS analysis. While mass spectrum provides the molecular weights of peptide fragments digested by protease such as trypsin, tandem mass spectrum produces amino acid sequence of digested peptides. Each amino acid sequences can be a query sequence in BLAST search to identify proteins. For the specimens of animals or plants of which genome sequences were known, we can easily identify expressed proteins from mass spectrums with high accuracy. However, for the other specimens such as ginseng, it is difficult to identify proteins with accuracy since all the protein sequences are not available yet. Here we compared the mass spectrums and the peptide amino acid sequences with ginseng expressed sequence tag (EST) DB. The matched EST sequence was used as a query in BLAST search for protein identification. They could offer the correct protein information by the sequence alignment with EST sequences. 90% of peptide sequences of ESI/Q-TOF/MS are matched with EST sequences. Comparing 68% matches of the same sequences with the nr database of NCBI, we got more matches by 22% from ginseng EST sequence search. In case of peptide mass fingerprinting from MALDI/TOF/MS, only about 19% (9 proteins of 47 spots) among peptide matches from nr DB were correlated with ginseng EST DB. From these results, we suggest that amino acid sequencing using tandem mass spectrum analysis may be necessary for protein identification in ginseng proteome analysis.

Flavonoid Biosynthesis: Biochemistry and Metabolic Engineering

Abstract: Flavonoid biosynthesis is one of the most extensively studied areas in the secondary metabolism Due to the study of flavonoid metabolism in diverse plant system, the pathways become the best characterized secondary metabolites and can be excellent targets for metabolic engineering These flavonoid-derived secondary metabolites have been considerably divergent functional roles: floral pigment, anticancer, antiviral, antitoxin, and hepatoprotective Three species have been significant for elucidating the flavonoid metabolism and isolating the genes controlling the flavonoid genes: maize (Zea mays), snapdragon (Antirrhinum majus) and petunia (Prtunia hybrida) Recently, many genes involved in biosynthesis of flavonoid have been isolated and characterized using mutation and recombinant DNA technologies including transposon tagging and T-DNA tagging which are novel approaches for the discovery of uncharacterized genes Metabolic engineering of flavonoid biosynthesis was approached by sense or antisense manipulation of the genes related with flavonoid pathway, or by modified expression of regulatory genes So, the use of a variety of experimental tools and metabolic engineering facilitated the characterization of the flavonoid metabolism Here we review recent progresses in flavonoid metabolism: confirmation of genes, metabolic engineering, and applications in the industrial use

In Vitro Selection and Characterizations of Gamma Radiation-Induced Salt Tolerant Lines in Rice

Abstract: The combination of radiation technique with an in vitro culture system was initiated to develop salt tolerant rice. We established an in vitro culture system to select tolerant lines against salt stress. NaCl tolerant cell lines were selected from the callus irradiated with gamma ray on N medium with 1.5% NaCl and 2 mg/L 2,4-D. Regenerants (M) were obtained from the tolerant callus which was cultured for 30 days auxin-free medium. The Mseeds were harvested from Mplants on an individual plant basis. Thirty seedlings from each 450 Mlines were transplanted in a field and total 5,000 Mlines were harvested with an average 90 percent of fertile grain. Mlines were utilized for selection of salt tolerance. Salinity-tolerant lines (225) were selected among 5,000 Mlines. Of the 225 lines tested, the morphological traits of two lines (120-10 and -11) were far superior to control (Donagjinbyeo) in agromomic traits such as plant height, root length and no. of roots. Control and tolerant lines were analyzed by RAPD markers. Three polymorphic bands were presented in only tolerant lines, demonstrating a genetic difference between control and the tolerant lines. Such tolerant lines could be used as genetic resources to improve salt tolerance.e.

Adventitious Root Formation from Cotyledon in Soybean (Glycine max L.) Cultivars

Abstract: The patterns of adventitious root formation from cotyledons for each cultivar of soybeans were compared. The results of adventitious root formation in cultivars are classified as two groups; the first group showed the direct adventitious root formation, and the second group resulted in the callus and adventitious root formation. The cultivars that have much callus formation had less the adventitious root formation. The adventitious root formation in the cotyledonary explants was occured only at the inoculation of adaxial side. When adaxial and abaxial side was inoculated simultaneously, the adventitious roots were formed at the adaxial side. Thus, it suggests that there must be direction to some extent. Starch in the cotyledonary explants were more abundant at the 4 days after induction than at the early stage of the adventitious root formation, but the starch was not observed after 7 days, that the growth stage of adventitious roots.

Plant Regeneration from Explant Types and Cultivars of Boxthorn (Lycium chinense Mill.)

Abstract: Callus and shoot formation of leaf explants from in vitro propagated shoots and field grown plants depending on the position of leaf, and four boxthron cultivars were investigated. Callus formation of explants from both in vitro shoot and field grown plants as easily achieved at the cut surfaces of explants but the callus formed from leaf of in vitro shoots was hardened as the duration of culture was proceed. Calli were effectively induced from leaves detached from the middle position of both in vitro and in vitro plants on MS medium containing 0.5 mg/L NAA with 0.2 mg/L BA, and the growth of calli were better in field grown leaves than in vitro grown leaves. Shoot formation were effectively induced from leaves detached from the upper position in vitro plants, and the middle parts of in vitro plants on MS medium containing 0.01 mg/L (NAA with 0.2 mg/ BA. There was difference on the frequency of shoot formation among four different cultivars; `Jindojaerae` was the best for shoot formation followed by `Cheonyang`, `Younghagukija` and `Cheongyangjaerae`.

Effects of Cultural Conditions on the In Vitro Propagation of Plantlets derived from Apical Meristem in Ipomoea batatas L.

Abstract: Sweet potato is a crop vegetatively propagated by vine cuttings, an ineffective method for maintaining pathogene-free stock plants. As an alternative method, single-node cultures of virus-free plantlets derived from apical meristem in sweet potato (cv. Yulmi) was examined. Effective pH range, sugar concentration and nodal order were investigated to establish an in vitro mass propagation system with high quality virus-free stock plantlets to farmhouse. Although the plantlets grew at wide range of pH, the most effective pH of the medium was 4.8 in single-node cultures. High sugar concentration of 60∼80 g/L resulted in increased growth response in shoot length, root length, number of node, leaf area and fresh and dry weight of shoot and root, whereas reducing sugar contents below 6% was showed reduced growth response. The first node including meristem tip was the best for the rapid growth of plantlets and the other nodes also showed a very similar growth response. Uniform plantlet can be obtained massively at the same time by culture of single node except for the first node including meristem tip. In conclusion, the most effective pH range and sugar concentration of medium for the growth of plantlets via single-node cultures was 4.8, 60∼80 g/L respectively. The first node was the best for the rapid propagation of plantlets in nodal order.

Effects of GA 3 and Charcoal on Plant Regeneration from Somatic Embryos of Acanthopanax sessiliflorus

Abstract: To establish the optimum condition for plant regeneration from somatic embryos of Acanthopanax sessiliflorus Rupr. et Maxim, a medicinal plant, somatic embryos were induced from zygotic embryo-derived embryogenic callus in hormoen-free MS medium. To induce plantlet conversion, cotyledonary somatic embryos were cultured on MS solid medium with GAat various concentrations (0~10 mg/L) for three weeks. Plantlets were transferred to 1/3 MS solid medium with 0.5% charcoal for 7 weeks. Stem length was increased proportionally to the concentration and treatment period of GA. Also, the highest leaf width (8.9 mm) and leaf number (2.84) of plantlet were obtained when plantlets were converted on 5,10 mg/L GApretreatments, respectively. The highest plant conversion frequency (66.7%) was obtained when the somatic embryos were cultured on medium containing 5 mg/L GA for 3 weeks and then were transferred to 1/3 MS medium with 0.5% charcoal. The highest survival rate of soil transfer was 90% when plantlets were regenerated on medium with 5 mg/L GA for 3 weeks and then transferred to plastic pots containing vermiculite and sand mixture for 4 weeks.

Growth Factors Affecting to Kalosaponins Contents of Kalopanax pictus Nakai

Abstract: Four kinds of kalosaponins from inner bark of Kalopanax pictus Nakai as a medicinal and edible timber tree species growing in Korea was analyzed by HPLC. A considerable range of variation in the contents was observed among population. The kalosaponins contents in inner bark from each population were the highest in the Mt. Barwang (30.37 mg/g on the dry weight basis) followed by Mt. Gariwang, Hanra II, Mangun, Paltan, and Hanra II population. Growth factors that might have influenced the contents of the kalosaponins were also examined. As both analysis results of partial correlation and multiple regression, the weight of inner bark and tree age were influenced the kalosaponin O contents. The kalosaponin B contents was influenced by both the weight of inner layer bark and annual mean DBH increment. The contents of kalosaponins appeared to be closely related to the inner bark characteristics.

Effect of Cytokinins on in Vitro Growth of Grapes (Vitis spp.)

Abstract: Effect of cytokinins (BA, TDZ, zeatin, 2iP, and kinetin) applied either singly or in combination on in vitro growth of two grape cultivars (`Cabernet Sauvignon` and `Campbell Early`) was investigated as a serial work for mass production of grapevine nursery stocks. In single treatment, shoot growth of two cultivars was most favorable in control. Shoot proliferation was satisfactory with 10 M BA regardless of cultivars and cytokinin combinations, followed by TDZ. Other treatments resulted in very poor or no branching. Total explants ready for subculture produced by 10 M BA outnumbered those by other treatments. TDZ was also effective. TDZ significantly increased the fresh weight and callus formation while shoot growth was unsatisfactory. Shoot growth response of two cultivars in combined treatments was also most favorable in control as was in single treatments. When TDZ was combined with zeatin, 2iP, and kinetin which failed to induce branching, proliferous branching was induced though the shoot number was behind that of single treatments of BA and TDZ. TDZ was very effective for total number of explants and fresh weight, showing 10-fold increase.

Multiple Shoot Formation from Cotyledonary Nodes of Soybean Cultivars

Abstract: For the plant regeneration of soybean (Glycine me L. Merr.), the shoot formation rate, optimal medium and tissue conditions were examined using Korean soybean cultivars. Among the parts of seedling, a node that includes one cotyledon showed the highest shoot formation rate among other tissues. Half-strength B5 medium was more efficient than full strength medium. Formation rates of pair shoots (1 to 2 shooting) were higher in the when benzyl adenine was supplemented. The formation rates of multiple shoots, that is, 4 to 5 in shooting, were high when thidiazuron was supplemented. Multiple shoot was de novo formed in cutting side of cotyledonary node. The effective concentration of thidiazuron for shoot induction treatment was 2 mg/L. Among the 27 cultivars, multiple shoot formation rates were high in the 11 cultivars including `Heugcheongkong, and pair shoot formation rates were high in the 16 cultivars including `Malikong`.